There have been a wide variety of attempts to develop technologies for producing a useful substance such as alcohols such as ethanol and butanol, and lactic acid by using as a raw material woody biomass such as wooden chips or soft biomass such as rice straw and bagasse.
Production of the useful substance from those biomass raw materials requires decomposition of the biomass raw materials to sugar and subsequent fermentation of the obtained sugar. In recent years, the biomass raw materials have been saccharified mainly by enzymes. However, the biomass raw materials include various impurities other than cellulose, and therefore, there is a problem in that the enzymes hardly act on the biomass raw materials. In this context, in order to enhance saccharification efficiency, there are developed various methods involving, as pretreatment, subjecting the biomass raw materials to steam treatment using dilute sulfuric acid, hot compressed water, or the like, treating the biomass raw materials with a supercritical ammonia fluid, and the like (for example, see Patent Literature 1).
In addition, there have been attempts to develop microorganisms useful for decomposition of the biomass raw materials.
For example, Patent Literature 2 discloses a method of improving decomposition capability for cellulose contained in the biomass raw materials by expressing a chimeric enzyme in which endocellulase (GH5) derived from Acidothermus cellulolyticus is fused with cellobiohydrolase (CBH) derived from Trichoderma reesei in Trichoderma reesei producing cellulase as a host.
Further, while it is necessary to conduct a saccharification step and a fermentation step independently for obtaining the useful substance such as ethanol from the biomass, there have been attempts to develop technologies capable of conducting these steps at once.
For example, Patent Literature 3 discloses providing a microorganism capable of directly hydrolyzing and fermenting lignocellulose-based biomass by enhancing activity of cellulase by modification. As one of the cellulases whose activity is to be enhanced, there are given hydrolase identified in Clostridium phytofermentans and the like, and a polynucleotide encoding the hydrolase and the like are disclosed.
In addition, Patent Literature 4 discloses a technology for expressing a chimeric protein including some cellulase derived from a microorganism not producing a cellulosome and a dockrin domain derived from a microorganism producing a cellulosome in yeast with a view to forming an artificial cellulosome. However, an artificial cellulosome is not formed in yeast in this literature.
The cellulosome is an enzyme complex formed of two components, i.e., a scaffolding protein (CbpA) not exhibiting enzyme activity and a group of cellulosomal enzymes. The scaffolding protein (CbpA) has a cellulose-binding domain (CBD), Cohesin domains, and Surface Layer Homology (SLH) domains. The group of cellulosomal enzymes have a dockerin domain to bind to the Cohesin domain of the scaffold protein (CbpA). Those components bind to each other to form the enzyme complex (cellulosome), and thereby, cellulose and hemicellulose can be degraded efficiently.
It can be said that microorganisms forming cellulosomes are useful microorganisms for decomposition and fermentation of the biomass raw materials. As such microorganisms, Clostridium cellulovorans (hereinafter sometimes referred to as C. cellulovorans)) and the like are known.
Clostridium cellulovorans (C. cellulovorans) is a gram-positive obligatory anaerobic mesophilic bacterium. The inventors of the present invention have confirmed that Clostridium cellulovorans (C. cellulovorans) has a genome size about 1 Mb larger than those of Clostridium cellulolyticum and Clostridium thermocellum belonging to the same genus Clostridium (see, for example, Non Patent Literature 1).
Moreover, Non Patent Literature 2 discloses that Clostridium cellulovorans (C. cellulovorans) produced useful substances such as acetate and butyrate by fermenting a test sample containing cellobiose and the like and also suggests that ethanol was produced.
Further, as a group of cellulosomal enzymes derived from Clostridium cellulovorans (C. cellulovorans), Endoglucanase E (EngE), which is endocellulase of Glycosyl Hydrolase family 5 (GH5), Endoglucanase K (EngK), which is processive endocellulase of Glycosyl Hydrolase family 9 (GH9), Endoglucanase H (EngH), which is processive endocellulase of Glycosyl Hydrolase family 9 (GH9), Exoglucanase S (ExgS), which is exocellulase of Glycosyl Hydrolase family 48 (GH48), and the like are known.
Of those, it is disclosed that EngK exhibits high activity for insoluble cellulose (see, for example, Non Patent Literature 3). In addition, it is also disclosed that each of a mixture of ExgS and EngE, a mixture of ExgS and EngH, and a mixture of EngE and EngH has specific activity for crystalline cellulose as compared to EngE, EngH, or ExgS alone (see, for example, Non Patent Literature 4).
As described above, it is suggested that Clostridium cellulovorans (C. cellulovorans) or an enzyme or group of enzymes derived from this microorganism are useful for decomposition and fermentation of the biomass.
However, the biomass such as woody biomass and soft biomass includes various impurities other than cellulose in accordance with the type of the biomass, unlike test samples, insoluble cellulose, crystalline cellulose, and the like. Therefore, direct decomposition and fermentation of the actual biomass (bagasse, rice straw, and the like) are not always possible.
Accordingly, there is a demand for a method enabling direct decomposition and fermentation of the actual biomass without the need to conduct a step of pretreatment and the like as well as the need to conduct the saccharification step and fermentation step independently. There is also a demand for a microorganism, an enzyme or group of enzymes, and the like capable of decomposing and fermenting the actual biomass in accordance with its type.